It has been a medical knowledge for over 100 years that nitroglycerine can be used in the treatment of coronary heart disease. It was only recognized 15 years ago, however, that this effect is attributable to the formation of nitric oxide (NO). Since then the significance of this molecule has been visible in an ever-increasing number of publications. Therefore ways of synthesizing NO, and particularly the discovery of compounds which modulate NO synthesis, are of great importance, particularly with regard to pharmacology.
NO is formed by NO synthases (EC: 1.14.13.39) (hereafter sometimes abbreviated to NOS). NOS catalyzes the oxidation of L-arginine to L-citrulline and NO via the intermediary NG-hydroxyarginine. NO synthases have a monomeric molecular mass of between 125 and 155 kDa, but are active only as homodimers. The molecular structure of the NO synthases strongly resembles that of the cytochrome P450 reductases. Various classes of NO synthases are known:
NOS inducible by cytokines or LPS (iNOS), and
constitutive NOS (cNOS), activated by Ca2+, which are further subdivided into the following subforms:                endothelial NOS=eNOS        neuronal NOS=nNOS.        
In the literature there are many examples of how to determine quantitatively the activity of known NOS isoenzymes. The following methods are the most well known:
1. Separation of the End Product Citrulline Using Column Chromatography

On completion of the reaction the enzyme mixtures are passed over separate cation exchange columns. Elution of the [3H]L-citrulline and determination of the eluates then takes place on a β-counter. The disadvantage of this method is that column chromatography has to take place separately for each enzyme mixture (e.g. 96 chromatography columns are needed per 96 well microtiter plate). See e.g., D. S. Bredt and S. H. Snyder, Proc. Natl. Acad. Sci. 87, 682 (1990).
2. NO2 Determination Using “Griess Reagent”
In the NOS reaction (see reaction 1) NO is obtained as an end product as well as L-citrulline. NO is, however, unstable and thus continues to react:2 NO+O2→2 NO22 NO2+H2O→NO2−+NO3−+2H+  (2)
The NO3− obtained must be reduced to NO2−. This can be achieved with cadmium or enzymatically by adding nitrate reductase. Since excess NADPH from the NOS mixture interferes with the Griess reaction with NO2−, these reduction products must be removed. This can be done enzymatically by adding lactate dehydrogenase/pyruvate. After addition of the Griess reagent (1% sulphanilamide: 0.1% naphthylenediamine dihydrochloride, 5% H3PO4) a purple-colored AZO dye is produced, which can be determined at 543 nm.
The Griess reaction takes place as follows:
(Green et al., Anal. Biochem., 126:131 (1982))
The disadvantage of this measuring method is that a plurality of separate reaction steps which must be carried out in sequence are necessary to determine NOS activity.
3. Measurement of Chemiluminescence
The NO obtained and L-citrulline in the NOS reaction react to form NO2− and NO3− (see reaction 2). However, to measure chemiluminescence NO2− and NO3− have to be converted back to NO. Then a reaction with ozone is carried out:NO+O3→NO2*+O2NO2*→NO2+hv  (4)(Archer, FASEB Journal, 7, 349 (1993)).
The disadvantage of this method is that a plurality of reaction steps are also needed. Furthermore, the generation of ozone is costly, which has to be produced particularly for the reaction because of its short half-life.
4. Determination of cGMP Using Guanylate Cyclase which can be Stimulated by NO.
Here the activity of guanylate cyclase can be determined from the quantity of cGMP. Since the, guanylate cyclase is activated by NO, the quantity of NO which is produced, and accordingly the activity of the NOS, can be extrapolated (Feelish et al., Eur. J. Pharmacol. 139:19 (1987); Mayer et al., Biochem. Biophys. Research Commun., 164:678 (1989)).
The disadvantage of this method is that it requires a coupled enzyme reaction. Coupled enzyme reactions usually do not permit the measurement of an initial rate because a clear linear determination of the reaction rate requires the substrates be present in concentrations close to enzyme saturation (˜100 Km). This can be a significant cost factor. Moreover, the activity of the guanylate cyclase is not constant in this reaction, as it is only stimulated by NO formed during the coupled reaction.
In modern pharmaceutical research, it is necessary to carry out a considerable number of measurements within the shortest possible time, in particular when testing large numbers of substances, known as libraries, so-called HTS processes (high throughput screening processes) are implemented to screen for individual substances with possible physiological effect. Test procedures used in HTS processes should be able to be automated, be as simple and as fast as possible and, in particular, only require a minimum of simple automatable process steps which do not require manual steps in between. A “rough method” delivering only a yes or no answer is sufficient, so measurements of linear initial rate of an enzyme reaction are not necessary. Time-consuming manipulations such as removal of aliquots, further incubations using coupled (enzyme) reactions, centrifugation steps which can not be automated etc., are automatically ruled out for an HTS operation. At the same time, however, the tests must provide reliable results. Therefore, the precision of the assay is of crucial significance for carrying out an HTS operation, i.e. the “signal to noise ratio” should be as high as possible, so that the precision of the result does not have to be won at the expense of two, three or four determination steps, which, in turn, hamper high volume throughput.
None of the reactions 1–4 presented above, thus, is suitable for HTS, because the coupled enzyme reactions, removal of aliquot parts, centrifugation steps, column chromatography separation steps etc. hamper high throughput screening. The time or number of process steps required to achieve the necessary precision is too great for an HTS process and the procedures are substantially not automatable.
The object of the present invention was, therefore, to develop a simplified method for measuring the activity of NO synthase, in particular also a method for identifying NO synthase modulators. This method should, in particular, be suitable for application in HTS.